1. Field of the Invention
This invention relates to improved reagents for use in CO.sub.2 analysis and to improved methods for determination of CO.sub.2 in body fluids using these reagents.
2. Description of the Prior Art
The determination of blood gases is important for clinical analysis. In particular, the determination of carbon dioxide (CO.sub.2) content is whole blood and blood serum are among the most frequently performed analyses in a clinical laboratory. Due to the great importance of these analyses, a number of techniques have been developed and they are presently being used to determine CO.sub.2 concentration.
A traditional technique used for the determination of CO.sub.2 in blood is the method of D. D. Van Slyke, which was published in the Journal of Biological Chemistry, Volume 61, p. 523 (1924). In the basic Van Slyke method, blood serum and acid are mixed in a closed vessel and the carbon dioxide in the blood is extracted from the blood by application of a vacuum. The extracted carbon dioxide is then measured volumetrically or manometrically. When the vacuum is drawn, other blood gases are released from the serum in addition to the carbon dioxide. This requires that a base, such as sodium hydroxide, be added in order to separate the carbon dioxide from the other released gases. After this, the volumetric measurement is performed by known techniques.
In order to overcome some of the disadvantages attendant to the practice of the Van Slyke method, Applicant herein has filed a copending application Ser. No. 524,793 (filed Nov. 18, 1974) now U.S. Pat. No. 3,964,864, which allows very rapid determination of CO.sub.2 content in body fluids. In that improved technique, an equilibrated substantially stationary system is used to obtain very accurate results using only very small samples. That apparatus is automatically cleaned after each measurement and a blood gas measured by the detector has essentially the same composition as that originally established in the reaction vessel in which the body fluid is mixed with the CO.sub.2 releasing reagent. Thus, that invention provides low cost, reliable measurement of CO.sub.2 without the need for vacuums and measurements are made under stationary equilibrium conditions. Only small sample volumes are required, and very quick measurements are made.
In the method of aforementioned Ser. No. 524,793, a small amount of body fluid sample is combined with a small amount of reagent in a closed vessel to release CO.sub.2 from the body fluid into the gas space of the vessel. Release of CO.sub.2 into the gas space is at atmospheric pressure. After the gas space has been equilibrated with the released gas, a displacement liquid (such as the reagent) is introduced into the reaction vessel, thereby pushing the equilibrated gas into a detector connected to the gas space of the reaction vessel. After the displacement of some of the gas from the gas space into the detector, the detector measures the concentration of a sample of CO.sub.2.
After the measurement is made, the mixture of sample and reagent in the closed vessel is drained from the vessel and excess flushing fluid (such as air) is passed through the detector and through the vessel in order to flush the apparatus prior to making another measurement.
Typically, the apparatus used to provide the improved method of CO.sub.2 analysis described in Ser. No. 524,793 (U.S. Pat. No. 3,964,864) comprises a reaction vessel which can be closed after the sample and reagent are introduced, a reagent means for putting reagent into the reaction vessel, a displacement means for adding fluid to the reagent vessel to push the released gas to a detector, and a flushing means for cleansing the system after each measurement. Typically, mechanical means such as a rotary slide valve is used to connect these various functional units into the system at the proper time.
The most frequently used acid for the release of CO.sub.2 from blood serum is lactic acid, in various concentrations such as 0.1M, 1.0M, or higher concentrations. Lactic acid such as this is used in particular as one of the preferred reagents in the apparatus of Ser. No. 524,793, now U.S. Pat. No. 3,964,864. Also, that application broadly suggests the use of detergents in the lactic acid.
For the best operation of apparatus for detection of CO.sub.2, it has been discovered that the reagent must meet several requirements other than the basic requirement of releasing CO.sub.2 from blood and blood serum. These other requirements are necessary for providing very high accuracy, precision, and speed for CO.sub.2 determinations in a practical system. Thus, experimentation has led to the use of the compositions described herein for release of CO.sub.2 from body fluids.
The requirements for preferred reagent compositions, which must be met in a practical system, include the following:
1. The CO.sub.2 should be released from the body fluid in a minimum of time so that fast analysis can be made.
2. The reagent should release CO.sub.2 from aqueous standard solutions (such as carbonate or bicarbonate solutions used for calibration) as fast as the reagent releases CO.sub.2 from blood serum. Thus, when ordinary lactic acid reagent is used, lower readings of CO.sub.2 will be obtained for aqueous solutions than for blood serum. In order to compensate for this, mixing times for aqueous solutions have to be longer in order to have complete release of the CO.sub.2 therein.
3. After the analysis is complete, the apparatus has to be drained of the body fluid, such as blood serum. The use of plain lactic acid reagent over many analyses often results in the accumulation of remains from the blood serum so that clogging of the lines eventually will occur. Therefore, an improved reagent is needed to promote complete drainage in the testing apparatus.
4. Mechanical components are used in the apparatus to perform CO.sub.2 analysis. These mechanical components are exposed to mechanical wear and tear which has to be minimized for long apparatus lifetime. For instance, slide valves often contain two flat plastic plates which rub on each other if there is no lubricant or gasket. In order to extend the life of the apparatus, the reagent should have lubricating properties.
5. The reagent solution should be chemically stable for long periods of time, preferably for many months.
6. A reagent solution should have a low solubility for CO.sub.2 in order to have large output signals and accurate results.
7. It is also desirable that the reagent be such that it will provide an indication of whether or not the CO.sub.2 measurement apparatus is performing properly. For example, the release of CO.sub.2 from standards and from serum requires that a certain minimum amount of acid reagent be present. If smaller amounts are present due to a malfunction of the apparatus, the results will be incorrect (i.e., too low). Thus, in the practice of the present invention, indicator dyes which are pH sensitive are used to provide an indication of a malfunctioning apparatus. For example, sodium carbonate standard solutions have a pH of about 11. When an excess of acid is added, the pH drops to about 3 or 4. A malfunction of the apparatus which adds only insufficient amounts of acid may leave the pH at 10 or 11. Thus, the indicator would provide a color indication that such malfunction was present.
The preceding seven requirements were obtained as a result of extensive experimentation to determine how to maximize operation of CO.sub.2 detectors whose operation is based on the Van Slyke technique. During this experimentation, many acids and additives were utilized. Some of these additives are more well known detergents, and each was tested with respect to the seven listed requirements. It was found that, while some of these modified reagents were satisfactory with respect to some of the requirements, only a limited class of acid reagents was suitable with respect to all of the requirements. This suitable class of reagents was not apparent from the use of surfactants, such as cleansers and detergents, in combination with an acid such as lactic acid. For example, many well known surfactants were combined with lactic acid and the resulting reagents tested, but these resulting reagents were found to be poor with respect to many of the performance standards. Thus, reagents comprising lactic acid and surfactants having the tradenames TWEEN-20, TWEEN-40, TWEEN-60, and TWEEN-80, available from J. T. Baker Chemical Company, Phillipsburg, New Jersey, have been found to yield poor results in this technique for CO.sub.2 measurement. Other surfactants which have not performed well for this specific purpose include surfactants having the tradenames Span-20, Span-40, Span-60, Span-65, Span-80, and Span-85, available from City Chemical Corp., New York, N.Y. Esters of various materials have been also utilized with rather poor results. Consequently, considerable experimentation was required to discover suitable reagents for use in releasing CO.sub.2 from body fluids, such as blood serum.
Accordingly, it is a primary object of the present invention to provide improved reagents for release of CO.sub.2 from body fluids.
It is another object of the present invention to provide improved reagents for releasing CO.sub.2 from body fluids, which reagents will release the CO.sub.2 quickly and will act as lubricants in the apparatus used for CO.sub.2 detection.
It is still another object of the present invention to provide improved reagents for release of CO.sub.2 from body fluids, where the reagents will release CO.sub.2 from aqueous standard solutions used for calibration as quickly as they will release CO.sub.2 from samples of body fluids.
It is a further object of the present invention to provide reagents for release of CO.sub.2 from body fluids, using reagents which enhance drainage in the apparatus used to detect CO.sub.2.
It is a still further object of the present invention to provide improved reagents for releasing CO.sub.2 from body fluids, where the reagents have lubricating properties.
It is another object of the present invention to provide an improved method for detecting CO.sub.2 in body fluids, using improved reagents.
It is another object of the present invention to provide reagents for release of CO.sub.2 from body fluids, which reagents provide an indication of apparatus malfunction in the apparatus used for such analysis.
It is a further object of this invention to provide improved reagents for release of CO.sub.2 from body fluids, where the reagents have a low solubility for CO.sub.2.